Emergence of NDM-producing Enterobacterales infections in companion animals from Argentina

Antimicrobial resistance is considered one of the most critical threat for both human and animal health. Recently, reports of infection or colonization by carbapenemase-producing Enterobacterales in companion animals had been described. This study report the first molecular characterization of NDM-producing Enterobacterales causing infections in companion animals from Argentina. Nineteen out of 3662 Enterobacterales isolates analyzed between October 2021 and July 2022 were resistant to carbapenemes by VITEK2C and disk diffusion method, and suspected to be carbapenemase-producers. Ten isolates were recovered from canine and nine from feline animals. Isolates were identified as K. pneumoniae (n = 9), E. coli (n = 6) and E. cloacae complex (n = 4), and all of them presented positive synergy among EDTA and carbapenems disks, mCIM/eCIM indicative of metallo-carbapenemase production and were also positive by PCR for blaNDM gene. NDM variants were determined by Sanger sequencing method. All 19 isolates were resistant to β-lactams and aminoglycosides but remained susceptible to colistin (100%), tigecycline (95%), fosfomycin (84%), nitrofurantoin (63%), minocycline (58%), chloramphenicol (42%), doxycycline (21%), enrofloxacin (5%), ciprofloxacin (5%) and trimethoprim/sulfamethoxazole (5%). Almost all isolates (17/19) co-harbored blaCTX-M plus blaCMY, one harbored blaCTX-M alone and the remaining blaCMY. E. coli and E. cloacae complex isolates harbored blaCTX-M-1/15 or blaCTX-M-2 groups, while all K. pneumoniae harbored only blaCTX-M-1/15 genes. All E. coli and E. cloacae complex isolates harbored blaNDM-1, while in K. pneumoniae blaNDM-1 (n = 6), blaNDM-5 (n = 2), and blaNDM-1 plus blaNDM-5 (n = 1) were confirmed. MLST analysis revealed the following sequence types by species, K. pneumoniae: ST15 (n = 5), ST273 (n = 2), ST11, and ST29; E. coli: ST162 (n = 3), ST457, ST224, and ST1196; E. cloacae complex: ST171, ST286, ST544 and ST61. To the best of our knowledge, this is the first description of NDM-producing E. cloacae complex isolates recovered from cats. Even though different species and clones were observed, it is remarkable the finding of some major clones among K. pneumoniae and E. coli, as well as the circulation of NDM as the main carbapenemase. Surveillance in companion pets is needed to detect the spread of carbapenem-resistant Enterobacterales and to alert about the dissemination of these pathogens among pets and humans.


Introduction
Antimicrobial resistance (AMR) is a growing and serious threat for both human and animal health.In recent years, AMR has been accelerated by the misuse and overuse of antimicrobials in human and veterinary medicine [1].Antimicrobials are commonly indicated prophylactically and therapeutically, and as growth promoters in animal production in some countries, but only for treatment of infections in humans and companion animals [2].Companion animals are often treated with the same or similar antibiotics that are used in human health, which poses a serious risk of selection and dissemination of pathogens and resistance mechanisms that can circulate between human and animal populations [2,3].This situation is aggravated by close contact between pets and their owners, which increases the risk of transmission between them [3,4].
Particularly, carbapenem-resistant Enterobacterales (CRE) are considered one of the most critical problems associated with AMR [5].Carbapenems are broad-spectrum β-lactam antibiotics used to treat serious infections caused by multidrug-resistant pathogens [6].Infections caused by CRE have a high health burden and represent a real diagnostic and therapeutic challenge in healthcare centers [5].While CRE have been associated with nosocomial infections in humans, there has been an increase of reports of these pathogens in veterinary medicine in recent years [7].It is considered that the dissemination of CRE in companion animals would be through two main ways [4]: (i) by zooanthroponosis, where the direction of transmission would go from humans, colonized by these pathogens, to pets; or (ii) due to contamination of environments, mainly veterinary facilities.
NDM and OXA-48 are the most widespread carbapenemases reported in companion animals [12].The first report of NDM date back to 2008 in the United States where NDM-1-producing Escherichia coli isolates were found in canine and feline samples [13].Hereafter, other carbapenemase-producing Enterobacterales recovered form pets were reported worldwide [4,10].In Argentina, sporadic cases of carbapenem-resistant Enterobacterales were described in a recent retrospective surveillance study however the molecular mechanisms were not characterized [14].To the best of our knowledge, this study represents the first molecular characterization of NDMproducing Enterobacterales of infections in companion animals from Argentina.
Genetic relatedness among the isolates was evaluated by XbaI-digested PFGE using a CHEF-DR ® III System (Bio-Rad Laboratories, California, U.S.) as previously described [19].The DNA fragments were resolved in a 1% agarose gel applying a switching time of 2.2 to 54.2 s and a voltage of 6V/cm for 20 h at 14 °C in 0.5X TBE buffer.Isolates of the same pulsetype were considered, by inference, to belong to the same sequence type (ST).bla NDM variants were confirmed in all isolates by Sanger Sequencing (ABI PRISM 3100 o 3730, Applied Biosystems, Massachusetts, U.S.) and bla CTX-M group by monoplex PCR.Primers NDM-in-F (5'-CTA TTT ACT AGG CCT CGC ATT-3') and NDM-in-R (5'-ATA AAA CGC CTC TGT CAC AT-3') were used for sequencing the entire bla NDM gene.

Biparental conjugation
Horizontal gene transfer of bla NDM was evaluated by biparental conjugation assay in two selected isolates from each species: M27649 and M27789 for K. pneumoniae, M27717 and M27987 for E. coli and M27828 and M27716 for E. cloacae complex.E. coli J53 (sodium azide resistant and gentamicin susceptible) was used as recipient strain.A ratio of 3:1 of donor:recipient strains were mixed on tryptic soy agar plates and incubated during 18 h at 35°C.Conjugation mix was resuspended in 1ml of physiological saline solution.Transconjugants were selected on tryptic soy agar plates supplemented with 200 µg/ml sodium azide plus 40 µg/ml gentamicin.Gentamicin was used for transconjugant selection because aminoglycoside resistance, mediated by 16S rRNA methyltransferases [21], and is generally co-linked with the bla NDM gene.Transconjugants were identified by conventional biochemical methods and MALDI-TOF, and the bla NDM -acquisition was evaluated by the agar diffusion disks test, synergism among EDTA and carbapenem disks, and confirmed by PCR.
K. pneumoniae was the most common species causing infections in companion animals in our collection, and contrary to E. cloacae and E. coli, this species harbors different variants of bla NDM gene.All nine K. pneumoniae strains harbors bla CTX-M-1/15 group ESBL gene while eight of them were also positive for bla CMY gene.K. pneumoniae M22738 belonging to clone A showed additional resistance to chloramphenicol, fosfomycin and minocycline compared with the other isolates of the same clone.It could be explained by the acquisition of extra plasmid/s coding for resistance to these drugs by this strain.ST15 CTX-M-1/15 K. pneumoniae lineage was previously reported in companion animals and humans in Portugal [22] and among other countries [23].K. pneumoniae ST15 clone was also reported to produce different carbapenemases, however bla OXA-48 was the common [23,24].In our collection, 5/9 K. pneumoniae isolates were ST15 and were recovered from four institutions.All five isolates harbors bla NDM-1 , bla CTX-M-1/15 plus bla CMY genes, confirming the association of this lineage with CTX-M-15 and CMY.K. pneumoniae ST273 clone was also associated as a carbapenemase-producer, even though an isolate harbouring bla NDM-1 plus bla IMP-4 was described previously [25,26].Here we detected two isolates with different PFGE-patterns belonging to ST273, both harbouring bla NDM-1 , but K. pneumoniae M28018 isolate carrying two alleles of the same gene, bla NDM-1 and bla NDM-5 .Some carbapenemases shows low hydrolytic activity against imipenem, being not a good marker to detect some carbapenemase-producer isolates [12].Considering veterinary AST-GN98 card for VITEK2 ® system has only imipenem but do not include meropenem, ertapenem, or temocillin (a good phenotypic marker for screening OXA-48-like carbapenemases), there is a chance that some carbapenemases, as OXA-48-like, were overlooked.This drawback emphasizes the necessity of updating the carbapenemase screening protocols for veterinary laboratories using automated AST systems [12].
ST11 is the founder ST of CC11, a single locus variant of the hyper-epidemic ST258 clone.KPC-2-producing K. pneumoniae ST11 has been reported to be the dominant clone in China, South Korea and Argentina, among other countries, causing human infections [19,27].In companion animals, ST11 has been reported in France as a producer of plasmid-borne ampC bla DHA-1 [28] and in Germany and France as OXA-48 producer [24,29].K. pneumoniae M27986 belongs to ST11 and harbors bla NDM-5 variant and the unique isolate without the plasmid-borne ampC bla CMY in this collection.Carbapenemase-producing K. pneumoniae ST29 has been reported sporadically causing human infections, but also in abattoir wastewater from Pakistan where even some strains harbors multiple carbapenemases [29].K. pneumoniae M27789 belonging to ST29 was recovered from a dog´s urine sample and was positive for bla NDM-5 , bla CTX-M-1/15 and bla CMY genes.In a recent work, we described clinical Enterobacterales coproducing multiple carbapenemases, where K. pneumoniae CC307 and CC11 were the dominant clones associated with KPC-2 plus NDM-1 or KPC-2 plus NDM-5 [19].In that report, K. pneumoniae ST11 (n = 22) and ST5995, a SLV-ST15 (n = 1) were detected, which evidences the circulation of these clones producing carbapenemases between humans and companion animals.
E. coli ST162 is a global virulent lineage that has been recovered from humans, environmental samples as well as from food, wild animals, and companion animals, and has been found associated with bla CTX-M and/or mcr-1 genes [30,31].However, a unique carbapenemase-producing E coli ST162 was reported and recovered from lymph nodes sample from a pygmy sperm whale (Kogia breviceps) [32].Here we report three bla NDM-1 -producing E coli ST162 recovered from cats (n = 2) and dog, and coproducing bla CTX-M-2 ESBL gene.All these three E coli ST162 isolates showed the same antimicrobial resistance profile and were recovered from different veterinary clinics.
E. coli ST457 has a broad host range and is a globally disseminated lineage, which has been detected in Oceania, America, Asia and Europe from humans, wild animals, companion animals and food samples, and was mainly associated with bla CTX-M-1 and bla CMY-2 [33].In China, E. coli NDM-1-producing ST457 isolates associated with hemorrhagic pneumonia in mink (Neogale species) were found [34].E. coli M27717 isolate belonging ST457 was recovered from bone sample of a dog and harbors NDM-1, CTX-M-2 and CMY β-lactamases.E. coli ST224 isolates has been frequently recovered in Brazil [35] and Australia [36] from animals for consumption and companion animals, harbouring bla CTX-M-55 or bla CTX-M-15 ESBLs, respectively.E. coli M27739 ST224 was recovered from urine from a cat and harbors NDM-1, CTX-M-1/15 and CMY β-lactamases.Finally, E. coli ST1196 isolates has been reported in different environments and hosts [30], producing different carbapenemases like OXA-48 in companion animals [24] and NDM-1 or KPC-2 in humans [37].M27948 E. coli isolate, belonging to ST1196, recovered from a cat was the only one without a bla CTX-M ESBL gene, but harbouring bla NDM-1 and bla CMY genes.In a retrospective and longitudinal study of carbapenemase-producing E. coli isolates recovered from humans samples in Argentina [38], the major lineages observed were ST10 and ST131, however and interestingly, isolates belonging to ST457 (n = 1), ST224 (n = 1) and ST1196 (= 2) were also detected.Nevertheless, human isolates were KPC-2-producers instead of NDM-1 as was observed in pets.To our knowledge, the E. coli ST162 lineage, the major on this study, has not been previously described in Argentina in carbapenemase-producing E. coli isolates.
Carbapenemase-producing E. cloacae ST171 clone has been mainly reported from human samples producing KPC, NDM and OXA-48 carbapenemases, however two isolates producing KPC-4 carbapenemase and recovered from dogs were reported [39].E. cloacae ST544 producing IMP-26 was reported as an epidemic clone in a tertiary hospital from China [40].E. cloacae ST286 and ST61 had been found from dog samples in France (https:// www.ebi.ac.uk/ ena/ brows er/ view/ SAMN2 45841 16) and environmental samples in Australia (https:// www.ebi.ac.uk/ ena/ brows er/ view/ SAMN1 01747 34), respectively but not associated with carbapenemases-production.Among the four E. cloacae described in this work, all of them harbors bla NDM-1 variant and coproduces CTX-M and CMY β-lactamases.Only two of the E. cloacae clones described here were previously reported in dogs, however, and to the best of our knowledge, this is the first description of NDM-producing E. cloacae complex isolates recovered from cats.All isolates described here presented resistance to β-lactams including, as expected, carbapenems and ceftazidime/avibactam, aminoglycosides, ciprofloxacin and trimethoprim/sulfamethoxazole, being colistin, tigecycline and fosfomycin the most active antibiotics.

Conclusion
We report here the emergence of NDM-producing Enterobacterales recovered from companion animals in Argentina.Isolates belonged to different species, being K. pneumoniae the most frequent NDM-producer pathogen, followed by E. coli and E. cloacae complex.Both, dissemination of carbapenem-resistant Enterobacterales clones and horizontal bla NDM gene transfer mechanisms had contributed to the emergence and spread of NDM among pet isolates in our country.The finding of two NDM variants, bla NDM-1 or bla NDM-5 , suggest the circulation of different plasmids, and raises the needs of characterization of these elements at molecular level.Based on all these data, we consider that it is necessary to strengthen the diagnosis, surveillance and control of carbapenem-resistant Enterobacterales in companion animals to prevent the dissemination of these mechanisms in the context of One Health.